Nucleic acid promoters

ABSTRACT

Disclosed is a method of detecting the presence of a nucleic acid target sequence of interest, the method comprising the steps of:  
     (a) adding first and second nucleic acid probes to a sample comprising the sequence of interest, so as to form a complex comprising three strands of nucleic acid, wherein the first probe comprises the full length sequence of a first strand of a double stranded promoter, the target sequence comprises an end part of a second strand of the double stranded promoter which is complementary to a part of the first strand, and the second probe comprises the rest of the second strand of the double stranded promoter which is complementary to a part of the first strand, such that a functional promoter is formed when the first probe is hybridised to both the target sequence and to the second probe;  
     (b) adding a polymerase which recognises the promoter, so as to cause the de novo synthesis of nucleic acid from the promoter present in the complex; and  
     (c) detecting directly or indirectly the de novo synthesised nucleic acid. Also disclosed is the complex formed in performance of the method defined above, and a kit for performing the method defined above.

FIELD OF THE INVENTION

[0001] The present invention relates to nucleic acid hybridisationprobes and complexes formed therefrom, their use in nucleic acidamplification and/or nucleic acid detection processes and to kitscomprising the probes and for forming said complexes. The presentinvention is particularly concerned with transcription and amplificationof hybridised nucleic acid probes such that sensitivity of hybridisationreactions is increased.

BACKGROUND OF THE INVENTION

[0002] All publications mentioned in this specification are incorporatedherein by reference.

[0003] Much research has been carried out on RNA polymerases, especiallybacteriophage RNA polymerases. Generally, bacteriophage RNA polymerasesare exceptionally active for in vitro transcription. This high levelactivity may be due in part to the fact that they are composed of asingle polypeptide chain and do not require a dissociating initiationfactor. These polymerases have been shown to be more active onsupercoiled templates although they are also very active on lineartemplates (Smeekens & Romano 1986 Nucl. Acids Res. 14, 2811).

[0004] Specifically, the RNA polymerase from the bacteriophage T7 hasbeen shown to be very selective for specific promoters that are rarelyencountered in DNA unrelated to T7 DNA (Chamberlin et al., 1970 Nature228,227; Dunn & Studier 1983 J. Mol. Biol. 166, 477). T7 RNA polymeraseis able to make complete transcripts of almost any DNA that is placedunder control of a T7 promoter. T7 RNA polymerase is a highly activeenzyme that transcribes about five times faster than does Escherichiacoli RNA polymerase (Studier et al, 1990 Methods Enzymol. 185, 60). Thesynthesis of small RNAs using T7 RNA polymerase has been describedwhereby sequences around the RNA polymerase promoter sequence are shownto be important in the reproducible improvement of yield of RNA produced(Milligan & Uhlenbeck, 1989 Methods Enzymol. 180, 51 and Milligan et al,1987 Nucl. Acids Res. 15, 8783-8798). Other RNA polymerases that havesimilar properties to T7 include those from bacteriophage T3 and SP6,the genes for which have all been cloned and the corresponding enzymesare commercially available.

[0005] A number of nucleic acid amplification processes are disclosed inthe prior art. Ore such process is polymerase chain reaction (PCR)disclosed in US 4683195 and 4683202. The PCR amplification process isvery well-known and successful. However PCR does have drawbacksincluding the need for adjusting reaction temperatures alternatelybetween intermediate (e.g. 50° C.-55° C.) and high (e.g. 90° C.-95° C.)temperatures involving repeated thermal cycling. Also, the time scalerequired for multiple cycles of large temperature transitions to achieveamplification of a nucleic acid sequence and the occurrence of sequenceerrors in the amplified copies of the nucleic acid sequence is a majordisadvantage as errors occur during multiple copying of long sequencetracts. Additionally, detection of the amplified nucleic acid sequencegenerally requires further processes e.g. agarose gel electrophoresis.

[0006] Alternative nucleic acid amplification processes that do utilizeRNA polymerases are disclosed in WO 88/10315 (Siska Diagnostics), EP329822 (Cangene) EP 373960 (Siska Diagnostics), U.S. Pat. No. 5,554,516(Gen-Probe Inc.), WO 89/01050 (Burg et al, WO 88/10315 (Gingeras et al,and EP 329822 (Organon Teknika), which latter document relates to atechnique known as NASBA. These amplification processes describe acycling reaction comprising of alternate DNA and RNA synthesis. Thisalternate RNA/DNA synthesis is achieved principally through theannealing of oligonucleotides adjacent to a specific DNA sequencewhereby these oligonucleotides comprise a transcriptional promoter. TheRNA copies of the specific sequence so produced, or alternatively aninput sample comprising a specific RNA sequence (U.S. Pat. No.5,554,516), are then copied as DNA strands using a nucleic acid primerand the RNA from the resulting DNA:RNA hybrid is either removed bydenaturation (WO 88/10315) or removed with RNase H (EP 329822, EP 373960& U.S. Pat. No. 5,554,516).

[0007] The annealing of oligonucleotides forming a transcriptionpromoter is then repeated in order to amplify RNA production.Amplification is thus achieved principally through the use of efficientRNA polymerases to produce an excess of RNA copies over DNA templates.The RNase version of this method has great advantages over PCR in thatamplification can potentially be achieved at a single temperature (i.e.isothermally). Additionally, a much greater level of amplification percycle can be achieved than for PCR i.e. a doubling of DNA copies percycle for PCR; 10-100 RNA copies per cycle using T7 RNA polymerase.

[0008] The processes described above all refer to methods whereby aspecific nucleic acid region is directly copied and these nucleic acidcopies are further copied to achieve amplification. The variabilitybetween various nucleic acid sequences is such that the rates ofamplification between different sequences by the same process are likelyto differ, thus presenting problems for example in the quantitation ofthe original amount of specific nucleic acid.

[0009] The processes listed above have a number of disadvantages in theamplification of their target nucleic acid; therefore, a list ofdesiderata for the sensitive detection of a specific target nucleic acidsequence is outlined below;

[0010] a) the process should preferably not require copying of thetarget sequence,

[0011] b) the process should preferably not involve multiple copying oflong tracts of sequence,

[0012] c) the process should preferably be generally applicable to bothDNA and RNA target sequences including specific sequences withoutdiscrete ends,

[0013] d) the signal should preferably result from the independenthybridisation of two different probes, or regions of probe, to a targetsequence,

[0014] e) the process should preferably include an option for detectionof hybridised probe without any additional steps.

[0015] A nucleic acid amplification process that fulfils the abovedesiderata is disclosed in WO 93/06240 (Cytocell Ltd). Two amplificationprocesses are described, one thermal and one isothermal. Both thethermal and isothermal versions depend on the hybridisation of twonucleic acid probes of which regions are complementary to the targetnucleic acid. Portions of said probes being capable of hybridising tothe sequence of interest such that the probes are adjacent orsubstantially adjacent to one another, so as to enable complementary armspecific sequences of the first and second probes to become annealed toeach other. Following annealing, chain extension of one of the probes isachieved by using part of the other probe as a template. Amplificationof the extended probe is achieved by one of two means; in the thermalcycling version thermal separation of the extended first probe iscarried out to allow hybridisation of a further probe, substantiallycomplementary to part of the newly synthesised sequence of the extendedfirst probe. Extension of the further probe by use of an appropriatepolymerase using the extended first probe as a template is achieved.Thermal separation of the extended first and further probe productsprovides templates for the extension of further first probe moleculesand the extended first probe can act as a template for the extension ofother further probe molecules.

[0016] In the isothermal version, primer extension of the first probecreates a functional RNA polymerase promoter that in the presence of arelevant RNA polymerase, allows for transcription of the probe sequenceproducing multiple copies of RNA. The resulting RNA is further amplifiedas a result of the interaction of complementary DNA oligonucleotidescontaining further RNA polymerase promoter sequences, whereuponannealing and extension of the RNA on the DNA oligonucleotide leads to afurther round of RNA. This cyclical process generates large yields ofRNA, detection of which can be achieved by a number of means.

SUMMARY OF THE INVENTION

[0017] In a first aspect the invention provides a complex comprisingthree strands of nucleic acid: a promoter strand, a promotercomplementary strand, and a target strand; wherein the promotercomplementary strand comprises the full length sequence of a firststrand of a double stranded promoter; the target strand comprises a partof a second strand of the double stranded promoter; and the promoterstrand comprises a part of the second strand of the double strandedpromoter which is complementary to a part of the first strand; whereinneither part of the second strand of the double stranded promoterpresent on the target strand or on the promoter strand is capable offorming a substantially functional promoter when hybridised to thepromoter complementary strand in the absence of the other part, butwherein a substantially functional promoter is formed when the promotercomplementary strand is hybridised to both the target strand and thepromoter strand.

[0018] The promoter strand (“PS”) and the promoter complementary strand(“CS”) are conveniently provided as a pair of respective “PS” and “CS”nucleic acid probes. The probes may comprise DNA, peptide nucleic acid(PNA), locked nucleic acid (LNA), (less preferably RNA) or anycombination thereof.

[0019] PNA is a synthetic nucleic acid analogue in which thesugar/phosphate backbone is replaced by a peptide-linked chain(typically of repeated N-(2-aminoethyl)-glycine units), to which thebases are joined by methylene carbonyl linkages. PNA/DNA hybrids havehigh Tm values compared to double stranded DNA molecules, since in DNAthe highly negatively-charged phosphate backbone causes electrostaticrepulsion between the respective strands, whilst the backbone of PNA isuncharged. Another characteristic of PNA is that a single base mis-matchis, relatively speaking, more destabilizing than a single base mis-matchin heteroduplex DNA. Accordingly, PNA is useful to include in probes foruse in the present invention, as the resulting probes have greaterspecificity than probes consisting entirely of DNA. Synthesis and usesof PNA have been disclosed by, for example, Orum et al, (1993 Nucl.Acids Res. 21, 5332); Egholm et al, (1992 J. Am. Chem. Soc. 114,1895);and Egholm et al, (1993 Nature 365, 566).

[0020] LNA is a synthetic nucleic acid analogue, incorporating“internally bridged” nucleoside analogues. Synthesis of LNA, andproperties thereof, have been described by a number of authors: Nielsenet al, (1997 J. Chem. Soc. Perkin Trans. 1, 3423); Koshkin et al, (1998Tetrahedron Letters 39,4381); Singh & Wengel (1998 Chem. Commun. 1247);and Singh et al, (1998 Chem. Commun. 455). As with PNA, LNA exhibitsgreater thermal stability when paired with DNA, than do conventionalDNA/DNA heteroduplexes. However, LNA can be synthesised on conventionalnucleic acid synthesising machines, whereas PNA cannot. Therefore, insome respects, LNA is to be preferred over PNA, for use in probes inaccordance with the present invention.

[0021] The substanially functional promoter created by the formation ofthe complex of the invention is an RNA promoter (i.e. a structurerecognised by an RNA polymerase and which causes the synthesis of RNA inthe presence of a suitable polymerase and reagents). A “substantiallyfunctional” promoter may be defined for present purposes as a nucleicacid complex which possesses at least 20% or more (preferably at least50%, more preferably at least 75%, and most preferably at least 90%) ofthe promoter activity of a fully double stranded, wild type promotersequence, the relative amount of promoter activity being measured byquantitation of the amount of a given RNA transcript produced by thepromoter in a given amount of time, under equivalent conditions (e.g. oftemperature and ribonucleotide triphosphate concentration).

[0022] The target strand may comprise any nucleic acid (RNA or, morepreferably DNA) sequence of interest, such as a sequence from a pathogen(such that the complex may be used to detect the presence of apathogen), or may be the sequence of a particular human, animal or plantallele, such that the genotype of an individual human or animal may bedetermined. Conveniently (but not necessarily) at least that portion(typically 2-4 bases) of the target which contains the part of thesecond strand of the double stranded promoter will preferably compriseDNA. The target strand may comprise both DNA and/or RNA.

[0023] In a second aspect the invention provides a method of detectingthe presence of a nucleic acid target sequence of interest, the methodcomprising: adding first and second probes to a sample comprising thesequence of interest, so as to form the complex of the first aspect ofthe invention; causing the synthesis of newly-synthesised ribonucleicacid from the substantially functional promoter present in the complex:and detecting directly or indirectly the newly-synthesised nucleic acid.The method may be used qualitatively or quantitatively. In particular,the method of the invention (and kits, as defined below) may be used fordetecting the presence of single nucleotide polymorphisms (“SNP”s) inthe target sequence, and may be used in high throughput screening (HTS)for pharmacogenomic investigations.

[0024] In a third aspect the invention provides a kit for forming thecomplex of the first aspect of the invention, the kit comprising a pairof probe molecules corresponding to the promoter strand and the promotercomplementary strand, and appropriate packaging means. The kit willpreferably be suitable for performing the method of the second aspect ofthe invention. The kit will therefore optionally comprise one or more ofthe following components: an RNA polymerase (particularly a T3, T7 orSP6 RNA polymerase), a DNA polymerase (particularly Klenow fragment ofDNA polymerase 1, f29 polymerase, Bst polymerase and Sequenase™),deoxyribonucleotide or ribonucleotide triphosphates (labelled orunlabelled), labeling reagents and/or detection reagents (e.g.fluorophores), buffers, and instructions for use according to the methodof the second aspect of the invention.

[0025] Thus, typically, the nucleic acid complex of the invention willcomprise a target sequence, a CS probe, and a PS probe. The CS probe(bearing the “promoter complementary strand”) comprises atarget-specific region (or “foot”) which hybridises specifically to thetarget sequence. This target-specific region comprises the first fewbases (preferably the first 2-4 bases, most preferably the first threebases) of an RNA polymerase promoter, which are hybridised withcomplementary bases in the target sequence. This “foot” region of the CSprobe may conveniently comprise LNA and/or PNA, which increases thespecificity of hybridisation. Where PNA is used, all or nearly all ofthe target complementary region may comprise PNA. If LNA is used, itwill normally suffice for 2-5 bases of the target-complementary portionto comprise LNA, the rest typically comprising conventional nucleicacid. The CS probe also comprises a non target-complementary “arm”region, which is adjacent to and contiguous with the target-specificfoot region and which comprises the rest of the RNA polymerase promotersequence.

[0026] The PS probe (bearing the “promoter strand”) comprises a portionthat is complementary to the “arm” region of the CS probe. The PS probeprovides the rest of the sequence required to form a substantiallyfunctional RNA polymerase promoter. If desired, the PS probe mayadditionally comprise a target-specific “foot” region which hybridisesto the target strand in a position substantially adjacent to the CSprobe, but the presence of such a target-specific region in the PS probeis not essential for performance of the invention. Where the PS probecomprises a target-complementary “foot”, the foot may comprise PNAand/or LNA, as described above.

[0027] The PS probe preferably comprises a 5′ template portion, which istranscribed into multiple RNA copies upon formation of the functionalRNA polymerase promoter. The general principle of the invention isillustrated in FIGS. 1 and 2, and described in greater detail below. Thearrangement is such that, in the absence of target nucleic acid,substantially no de novo RNA is synthesized, as no substantiallyfunctional RNA promoter is formed (the probes hybridised together, inthe absence of target, being unable to provide at least 20% of theactivity of the fully double stranded wild type promoter).

[0028] The present inventors are the first to appreciate that one of thestrands of a double stranded RNA promoter may be discontinuous, andformed by non-ligated separate nucleic acid molecules, and yet stillprovide a substantially functional RNA promoter. More particularly, theinventors are the first to appreciate that this phenomenon can beutilised to provide a method of detecting the presence and/or amount ofa nucleic acid sequence of interest.

[0029] The RNA polymerase promoter is preferably one recognised by abacteriophage RNA polymerase, for example, T3, T7 or SP6 polymerase orany of the mutant forms thereof which are known to those skilled in theart. Particular mutant RNA polymerases which may be useful in performingthe method of the invention are known, which may synthesise RNA or DNA(see Kostyuk et al, 1995 FEBS Letts. 369, 165-168).

[0030] The sequence of the T3 RNA polymerase promoter (described in theprior art) is: 5′ AAATTAACCCTCACTAAA 3′ (Seq. ID Nos. 1 and 2)3′ TTTAATTGGGAGTGATTT 5′

[0031] (A number of variant T3 promoter sequences are also known,especially those in which the first three bases of the non-templatestrand [the upper strand shown above] are 5′TTA 3′, rather than AAA.)

[0032] The sequence of the T7 RNA polymerase promoter (described in theprior art) is: 5′ TAATACGACTCACTATA 3′ (Seq. ID Nos. 3 and 4)3′ ATTATGCTGAGTGATAT 5′

[0033] The sequence of the SP6 RNA polymerase promoter (described in theprior art) is: 5′ ATTTAGGTGACACTATA 3′ (Seq. ID Nos. 5 and 6)3′ TAAATCCACTGTGATAT 5′

[0034] It is desirable that at least one of the probes in the complexcomprises a “template portion” which may be used as a template by apolymerase which recognises the promoter formed in the complex, suchthat the formation of the complex of the invention can allow for thesynthesis of newly-synthesised ribonucleic acid, which can be detecteddirectly or indirectly in any of a number of ways which will be apparentto those skilled in the art. The template portion is advantageouslypresent on the promoter strand.

[0035] It will generally be preferred for the 3′ end of the promoterstrand to be blocked in some way, so that RNA polymerase-mediatedextension thereof is not possible. This is especially desirable wherethe promoter strand comprises a target complementary portion. Blockingof the 3′ end is conveniently accomplished by providing a phosphategroup, or a propyl group, instead of an —OH group, on the 3′ terminalnucleotide. Other methods of blocking the 3′ end are well known to thoseskilled in the art.

[0036] The present inventors have found that the efficiency ofinitiation of RNA synthesis by the RNA polymerase promoter is affectedby sequences adjacent to the promoter, downstream. In particular, aregion of twelve bases (the “+12 region”) is required for optimum RNAtranscription. It is therefore preferred that the template portion ofthe complex, which is transcribed, comprises a +12 region appropriate tothe polymerase which recognises the promoter. The inventors haveelucidated the optimum sequence of +12 regions for the T7 polymerase(discussed in greater detail below)—it is not known at present if theseare also optimum for, say, T3 and SP6polymerases. If, as is possible,SP6 and T3 polymerases have different optimum +12 regions, it would be asimple matter for the person skilled in the art to identify the relevantsequence by trial-and-error, with the benefit of the present disclosure.

[0037] The sequences of preferred +12 regions, for inclusion in thetemplate portion of the promoter strand, (in respect of T7 polymerase)are shown below in Table 1. The most active +12 region (giving greatesttranscription) is at the top, with the other sequences shown indecreasing order of preference. TABLE 1 Alternative template +1 to +12sequences for T7 polymerase, in descending order of transcriptionefficiency (Seq. ID Nos. 7-15 respectively). 5′ GTTCTCTCTCCC 3′5′ GCTCTCTCTCCC 3′ 5′ GTTGTGTCTCCC 3′ 5′ GATGTGTCTCCC 3′ 5′ ATCCTCTCTCCC3′ 5′ GTTCTCGTGCCC 3′ 5′ ATCCTCGTGCCC 3′ 5′ GCTCTCGTGCCC 3′5′ GTTGTGGTGCCC 3′

[0038] In a further embodiment, the template portion of the complex(preferably on the promoter strand) could contain sequences that can beused to identify, detect or amplify the de novo synthesised RNA copies(see, for example, WO 93/06240, U.S. Pat. No. 5,554,516, or, forexample, using molecular beacon sequences such as those disclosed byTyagi & Kramer 1996 Nature Biotech 14, 303-308). These sequences areconveniently placed adjacent to, and downstream of, a +12 region (asdescribed above) and may comprise, but are not limited to, one or moreof the following: unique “molecular beacon” sequences; capturesequences; detection probe complementary sequences; alternative RNApromoter sequences for use in an isothermal amplification cyclingreaction (see below). A particular unique sequence especially useful inthe present invention is provided by bases 791-820 of 16S ribosomal RNAfrom Streptomyces brasiliensis (Stackebrandt et al, 1991 Appl. Environ.Microbiol. 57, 1468-1477), which sequence has no alignment with anyknown human DNA or DNA of a known human pathogen.

[0039] In a further embodiment of the invention it may be advantageous,when seeking to detect a sequence of interest in a mixture comprisingdouble stranded DNA (such as genomic DNA), to include in thehybridisation mixture one or more of further oligonucleotides (“blockingoligonucleotides”). These blocking oligonucleotides (preferably providedas a pair) bybridise to the sequence of interest, typically on each sideof the portion which is complementary to the first probe (and theportion complementary to the second probe, if the second probe comprisesa target-complementary portion). The blocking oligonucleotidespreferably comprise DNA, PNA, LNA (or a combination thereof) andadvantageously each comprise at least 10 (more preferably at least 20)nucleotides. The purpose of the blocking oligonucleotides is to inhibit(under the hybridisation conditions employed) re-annealing of the targetstrand with its complementary strand. The blocking oligonucleotides mayanneal to the target strand substantially adjacent to the first andsecond probes, or may anneal at a distance (e.g. 5-50 bases) therefrom.

[0040] Blocking oligonucleotides may offer little advantage if the firstand/or second probes contain large target-complementary “feet” regions.

[0041] Detection Methods

[0042] RNA produced in accordance with the method of the invention couldbe detected in a number of ways, preferably following amplification(most preferably by means of an isothermal amplification step). Forexample, newly-synthesised RNA could be detected in a conventionalmanner (e.g. by gel electrophoresis), with or without incorporation oflabelled bases during the synthesis.

[0043] Alternatively, for example, newly-synthesised RNA could becaptured at a solid surface (e.g. on a bead, or in a microtitre plate),and the captured molecule detected by hybridisation with a labellednucleic acid probe (e.g. radio-labelled, or more preferably labelledwith an enzyme, chromophore, fluorophore and the like).

[0044] One preferred detection method involves the use of molecularbeacons or the techniques of fluorescence resonance energy transfer(“FRET”), delayed fluorescence energy transfer (“DEFRET”) or homogeneoustime-resolved fluorescence (“HTRF”). Molecular beacons are moleculeswhich a fluorescence signal may or may not be generated, depending onthe conformation of the molecule.

[0045] Typically, one part of the molecule will comprise a fluorophore,and another part of the molecule will comprise a “quencher” to quenchfluorescence from the fluorophore. Thus, when the conformation of themolecule is such that the fluorophore and quencher are in closeproximity, the molecular beacon does not fluoresce, but when thefluorophore and the quencher are relatively widely-separated, themolecule does fluoresce. The molecular beacon conveniently comprises anucleic acid molecule labelled with an appropriate fluorophore andquencher.

[0046] One manner in which the conformation of the molecular beacon canbe altered is by hybridisation to a nucleic acid, for example inducinglooping out of parts of the molecular beacon. Alternatively, themolecular beacon may initially be in a hair-pin type structure(stabilised by self-complementary base-pairing), which structure isaltered by hybridisation, or by cleavage by an enzyme or ribozyme.

[0047] FRET (Fluorescence Resonance Energy Transfer) occurs when afluorescent donor molecule transfers energy via a nonradiativedipole-dipole interaction to an acceptor molecule. Upon energy transfer,which depends on the R⁻⁶ distance between the donor and acceptor, thedonor's lifetime and quantum yield are reduced and the acceptorfluorescence is increased or sensitised.

[0048] The inventors have used FAM (6-carboxyfluorescein) and TAMRA(N,N,N′,N′-tetramethyl-6-carboxy rhodamine) as donor and acceptor in anucleic acid hybridisation assay. The assay uses two dye labelled DNAoligomers (15 mers). FAM is linked to the 5′ of one probe and TAMRA tothe 3′ of the other. When hybridised to target nucleic acid the probesare positioned adjacent to one another and FRET can occur. Theinventors' experiments have demonstrated that for maximum signal theprobes need to be spaced by five bases.

[0049] Another approach (DEFRET, Delayed Fluorescence Energy Transfer)has been to exploit the unique properties of certain metal ions(Lanthanides e.g. Europium) that can exhibit efficient long livedemission when raised to their excited states (lexcitation=337 nm,lemission =620 nm). The advantage of such long lived emission is theability to use time resolved (TR) techniques in which measurement of theemission is started after an initial pause, so allowing all thebackground fluorescence and light scattering to dissipate. Cy5 (AmershamPharmacia) (lexcitation=620 nm, lemission=665 nm) can be used as theDEFRET partner.

[0050] HTRF (see WO92/01224; U.S. Pat. No. 5,534,622) occurs where thedonor (Europium) is encapsulated in a protective cage (cryptate) andattached to the 5′ end of an oligomer. The acceptor molecule that hasbeen developed for this system is a protein fluorophore, called XL665.This molecule is linked to the 3′ end of a second probe. This system hasbeen developed by Packard.

[0051] In another embodiment, the newly-synthesised RNA, before or afteramplification, results in formation of a ribozyme, which can be detectedby cleavage of a particular nucleic acid substrate sequence (e.g.cleavage of a fluorophore/quencher dual-labelled oligonucleotide).

[0052] Amplification Techniques

[0053] In preferred embodiments of the present invention, the RNAderived from the target dependent transcription reaction is amplifiedprior to detection, the amplification step typically requiring theintroduction of a DNA oligonucleotide. The amplification step isadvantageously effected isothermally (i.e. without requiring thermalcycling of the sort essential in performing PCR). The introduced DNAoligonucleotide is complementary to the 3′ region of the newlysynthesised RNA and also contains the sequence of an RNA polymerasepromoter and a unique transcribable sequence (template portion). Uponhybridisation of the newly-synthesised RNA with the DNA oligonucleotide,a primer extension reaction from the 3′ end of the RNA, mediated by anadded DNA polymerase, produces a functional double stranded RNApolymerase promoter. In the presence of the relevant RNA polymerase,multiple copies of a second RNA species are synthesised from the uniqueregion of the DNA oligonucleotide. This RNA in turn can act as primer toa further round of primer extension and RNA synthesis. The synthesis offurther RNA requires the presence of another DNA oligonucleotide that iscomplementary to the 3′ region of the second RNA species. This DNAoligonucleotide also contains the sequence of an RNA polymerase promoterelement together with a sequence upon transcription of which producesRNA comprising sequences identical to that derived in the targetdependent transcription reaction. The 3′ end of the RNA thus synthesisedis complementary to the first DNA oligonucleotide and hence a cyclicalamplification system is generated (see FIG. 3).

[0054] In these embodiments, it is important that the RNA promoter(s)formed during the amplification step(s) is (are) selected to berecognised by a polymerase different to that which recognises the splitpromoter formed initially at the 2½ or 3 way junction, so as to avoidinadvertent formation of a complete promoter ab initio, which would givea very high background signal.

[0055] In a variant of the embodiment described above, the introducedDNA oligonucleotide hybridises to the de novo synthesised RNA, therespective sequences being such that a further RNA polymerase promoteris directly formed without the need for a DNA polymerase-mediatedextension step (see FIG. 13). A cycling reaction may then be performedessentially as described above, with the transcipt from one reactionhybridising with a DNA oligonucleotide to form a second RNA promoter,which produces a transcript comprising a sequence common to the originaltranscript.

[0056] In a further variant, an RNA species produced from a splitpromoter in turn comprises the first few bases of an RNA polymerasepromoter, such that the RNA may in turn be the target sequence for theformation of a second split promoter (at a 2½ or 3 way junction),leading to synthesis of a further RNA species. If desired the sequencesof the template portions can be selected so as to create anamplification cycle in which the RNA transcript from one split promoterforms the target for the creation of a second split promoter, whichproduces a transcript which re-forms the first split promoter. Thescheme is illustrated schematically in FIG. 15. It will be appreciatedthat, in an amplification cycle of this sort, there is no requirement touse a different RNA promoter sequence to that in the original 2½ or 3way junction, because the method would not create a fullydouble-stranded RNA promoter ab initio, in the absence of target.

[0057] The above system could be arranged such that an RNA transcriptcomprised a plurality of portions of RNA promoters, so as to be capableof forming a plurality of split promoters in a single cycle, therebyincreasing the amount of amplification. Similarly, in the other types ofamplification cycles described above, the added oligonucleotides could,if desired, be capable of forming a plurality of RNA promoters.

[0058] In the above amplification strategies, some background “noise”may be created because of the tendency of many RNA polymerases (atrelatively low frequency) to produce RNA transcripts of a singlestranded DNA sequence such that, for example, referring to FIG. 3, sometranscription of DNA oligonucleotides (16) and (22) may occur even inthe respective absence of RNA molecules (14) and (20); or, the samephenomenon may occur, with reference to FIG. 13, in the absence of RNAmolecules (14) and (52). It is possible that this low level ofbackground transcription can be reduced by designing the DNAoligonucleotides (16 and 22 in FIG. 3; 50 and 54 in FIG. 13) so as toincorporate near their 3′ end a sequence which tends to causetermination of transcription. One example of such a sequence, which isespecially effective at terminating T7 polymerase-mediatedtranscription, is AACAGAT (in the template strand), as disclosed by Heet al, (1998 J. Biol. Chem. 273, 18,802). The same or a similartermination sequence could be positioned at the 5′ end of the DNAtemplate to increase processivity.

[0059] Various embodiments of the invention will now be described by wayof illustrative examples and with reference to the accompanyingdrawings, in which:

[0060]FIG. 1 is a schematic representations of a complex in accordancewith the invention, comprising a “three way” junction;

[0061]FIGS. 2, 4, 6-9, 11 and 12 are schematic representations of acomplex in accordance with the invention, comprising a “2½ way”junction;

[0062]FIGS. 3, 13 and 15 are schematic representations of a method ofdetecting a target sequence of interest by amplifying nucleic acidsynthesis;

[0063]FIG. 5 is a bar chart showing relative fluorescence units presentfollowing various nucleic acid amplification reactions; and

[0064]FIGS. 10 and 14 are bar charts showing picomoles of RNA producedfollowing various nucleic acid amplification reactions.

[0065]FIG. 1 shows a complex in accordance with the invention. Thecomplex comprises a promoter complementary strand “CS” (2), a promoterstrand “PS” (4), and a target strand (6). The CS (2) comprises the fulllength sequence of a first strand of a double stranded promoter (marked“Pr” in the figure). The target strand (6) comprises three bases whichare an end part (8) of a second strand of the double stranded promoterwhich is complementary to part of the CS (2). The PS (4) comprises therest of the second strand of the double stranded promoter, which part iscomplementary to the first strand of the promoter provided on the CS(2). Hybridisation of the CS (2) to the target strand (6), orhybridisation of the CS (2) to the PS (4), is not sufficient toconstitute a functional, double stranded promoter. However, asubstantially functional promoter is formed upon hybridisation of the CS(2) with both the target strand (6) and the PS (4), which represents acomplex in accordance with the present invention.

[0066] In the embodiment shown in FIG. 1, the PS (4) comprises a portion(10) which is complementary to the target strand (6), such that thecomplex forms what may be described as a “three way junction”. In analternative embodiment, illustrated schematically in FIG. 2, the PS (4)does not comprise a portion complementary to the target strand (6), suchthat the complex forms what may be described as a “two-and-a-half wayjunction” (2½ way junction).

[0067] In both of the embodiments illustrated in FIGS. 1 and 2, the PS(4) comprises a template portion (12), which can act as a templatenucleic acid strand for de novo nucleic acid synthesis once thefunctional promoter has been formed. Template portion (12) alsopreferably comprises a +12 region to optimise efficiency oftranscription by the RNA polymerase. The newly-synthesised nucleic acidis conveniently RNA, synthesised under the influence of an RNApolymerase promoter, such that multiple RNA transcripts (14) of thetemplate portion (12) are formed.

[0068] The de novo synthesised nucleic acid (14) may be detecteddirectly or indirectly. Preferably the de novo synthesised nucleic acid(14) is subjected to an amplification process prior to detection. Alarge number of suitable detection methods will be apparent to thoseskilled in the art. For example, the de novo synthesised nucleic acid(14) might hybridise to a complementary oligonucleotide molecular beaconsequence (e.g. as described by Tyagi & Kramer, 1996 Nature Biotechnology14, 303-308), such that de novo nucleic acid synthesis leads to anincrease, or a decrease as appropriate, in a fluorescence signal.Alternatively, the template portion (12) may be appropriately selectedsuch that DNA or RNA molecules synthesised with the portion (12) as atemplate may comprise, for example, capture sequences or detectionsequences.

[0069] As mentioned above, the de novo synthesised nucleic acid ispreferably subjected to an amplification step prior to detection. Theamplification step is such that a small amount of de novo synthesisednucleic acid results in the generation of a large amount of signal.Desirably, the amplification step is accomplished by performing two ormore nucleic acid synthesis steps in a cyclical manner, such that thenucleic acid product of a first synthesis step acts as the primer for asecond nucleic acid synthesis step, the product of which acts as theprimer for the first nucleic acid synthesis step, and so on. Cyclingamplification of this sort is disclosed in WO93/06240.

[0070]FIG. 3 is a schematic representation of an embodiment of acyclical nucleic acid synthesis, resulting in nucleic acidamplification. In FIG. 3, the 3′ end of a de novo synthesised RNAtranscript (14) produced from the template portion (12) of the secondprobe (4), is hybridised to an added DNA oligonucleotide (16). In step(i) the 3′ end of the transcript (14) is extended by the addition ofribonucleotides and/or deoxyribonucleotides in the presence of anappropriate polymerase. In the illustrated embodiment the extendedportion (of the transcript (14)) is of course complementary to theoligonucleotide (16) and forms an active double stranded RNA promoter(18) which is recognised by the appropriate RNA polymerase, so as toproduce multiple copies of a second RNA species (20) which is atranscript of the 5′ end of the DNA oligonucleotide (16).

[0071] In turn, the 3′ end of the RNA molecules (20) can hybridise to afurther added DNA oligonucleotide (22) (step (ii)). As previously, the3′ end of the RNA molecule (20) can undergo primer extension (step iii)by the addition of ribo- or (preferably) deoxyribonucleotides, therebyforming an active double stranded RNA promoter (24), which is recognisedby the relevant RNA polymerase which produces multiple copies of an RNAmolecule which is a transcript of the 5′ end of the DNA oligonucleotice(22).

[0072] The sequence of the DNA oligonucleotides (16) and (22) ispreferably selected such that the RNA transcripts produced from theoligonucleotide (22) comprise sequences which are identical to thosepresent in the RNA transcripts (14) produced originally, such that acycle is formed (step iv), in which the most recently synthesised RNAmolecules can hybridise to DNA oligonucleotide (16), be extended to formthe RNA promoter (18) and so on. In this way, massive amplification ofthe original transcript (14) may be achieved, thereby greatly enhancingthe sensitivity of the detection method of the invention.

[0073]FIG. 15 is a schematic representation of an amplification cycle inwhich a de novo synthesised RNA transcript (14), from a split promoterformed by the presence of the sequence of interest, hybridises to firstand second probes (60, 62 respectively) to form a second split promoter(indicated generally at 64). The sequence of the template portion ofsecond probe (62) is such that the RNA transcript (66) from splitpromoter (64), can act as target for a further pair of first and secondprobes (68, 70 respectively) creating a third split promoter (indicatedgenerally at 72). The sequence of the template portion of the secondprobe (70) is such that the RNA transcript produced by split promoter(72) has substantially the same sequence as the original RNA molecule(14), so that the second split promoter (64) can be reformed, therebycreating an amplification cycle.

EXAMPLES Example 1 Transcription from a Split T7 RNA Polymerase Promoterat a 2½ Way Junction.

[0074] This example demonstrates the creation of a functional DNAdependent RNA polymerase promoter as a result of the formation of a 2½way junction comprising target nucleic acid (Target: wild type human DNAcystic fibrosis transmembrane conductance regulator gene (CFTR) in whicha deletion of TTT causes a cystic fibrosis-encoding mutation DF508), apartly complementary oligonucleotide (complementary strand) and apromoter strand. In the example, the target sequence is provided by asynthetic oligonucleotide, which serves to demonstrate the principle ofthe invention. In practice, the target sequence would comprise a complexmixture of chromosomal DNA.

[0075] The example is illustrated schematically in FIG. 4. The completeT7 promoter is located towards the 3′ end of promoter complementarystrand probe (2). The first three (5′) bases of the promoter sequenceare complemented by three bases (3′ATT 5′) (8), in target strand (6),and probe (2) hybridises to the target (6) in such a way that the3′TTT5′ in the wild type is 14 bases downstream from the start of thepromoter. Hybridisation of a promoter strand probe (4), (at the 3′ endof which is the complement to the T7 promoter minus three bases) toprobe (2) forms a double stranded promoter, made complete by the threebases (8) in target (6), and therefore a split promoter is formed toyield a de novo synthesised RNA (14) in the presence of T7 RNApolymerase. For convenience, the promoter strand probe (4) is referredto hereafter as the PS probe, and the promoter complementary strandprobe (2) is referred to hereafter as the CS probe.

[0076] 1.1 Preparation of Oligonucleotides

[0077] The target oligonucleotides and probes were synthesised byphosphoramidite chemistry using an Applied Biosystems 380A synthesiser,used according to the manufacturer's instructions. All oligonucleotideswere HPLC purified using standard techniques.

[0078] 1.2 Split Promoter Probe and RNA Synthesis

[0079] Hybridisation reactions comprised mixtures of DNA includingtarget oligonucleotide (6), PS and CS probes, together with relevantcontrols comprising mixtures with and without target/probes. Forhybridisation reactions, 40 fmol of target oligonucleotide was mixedwith 40 fmol of PS probe and 40 fmol of CS probe in a solutioncontaining 4 μl 5× T7 RNA polymerase buffer (from Promega, giving 1×concentrations of 40 mM Tris (pH7.9), 6 mM MgCl₂, 2 mM spermidine and 10mM NaCl) and distilled water to a final volume of 20 μl (following finaladdition of T7 RNA polymerase and rNTP mix).

[0080] In this example, and others in the present specification,Milligan's buffer (Milligan et al, 1987 Nucl. Acids Res. 15, 8783-8798)may be used in place of Promega RNA polymerase buffer. Indeed, in thoseexamples where no DNA polymerase (e.g. where there is no DNApolymerase-dependent primer extension amplification step) is used,Milligan's buffer may be preferred. The composition of Milligan's bufferis as follows: 20 mM MgCl₂, 5 mM DTT, 80 mg/ml PEG, 50 μg/ml BSA, 0.01%(v/v) Triton X-100, 1 mM spermidine, and 40 mM Tris HCl, pH8.1.

[0081] The mixture was heated to 90° C. for 3 minutes to denature thenucleic acids, incubated on ice for 2 minutes, and equilibrated to 37°C. for 1 minute. Probes were annealed and transcribed at 37° C. for 180minutes by addition of 40 units of T7 RNA polymerase (Promega) and 40nmoles rNTP mix (Pharmacia Biotech). DNA oligonucleotides were removedfrom the reaction mix by the addition of 4 units of DNase I (Ambion) andincubating at 37° C. for 20 minutes prior to end detection. Theresulting product was immobilised by hybridisation to a specificbiotinylated oligonucleotide (probe 3) which was in turn bound to astreptavidin coated well. The immobilised product was detected by timeresolved fluorescence via the hybridisation of probe 4, a europiumlabelled oligonucleotide probe (see below).

[0082] 1.3 Detection of RNA by Time Resolved Fluorescence (TRF)

[0083] 5 μl of reaction sample was added to the reaction mix consistingof 145 μl of Wallac (E.G. & G. Wallac, Crown Hill Business Centre,Milton Keynes, UK) assay buffer, 0.9 pmol of probe 3 and 0.3 pmol ofprobe 4 in a well of a Labsystems streptavidin coated microtitre plate,which was incubated at room temperature for 60 minutes (N.B. the use oflonger probe PS gives a transcript with a longer capture tail, so thatcapture of this with the extended biotinylated probe 3a results in moresensitive detection). Unbound material was removed by washing the wells4× with 200 μl of Wallac wash solution. 180 μl of Wallac enhancementsolution was added to dissociate the europium from its chelated bondingto probe 4, and TRF was measured every 10 minutes up to 60 minutes usingthe europium protocol on a Wallac Victor 1420 Multilabel Counter. Theresults obtained (using Probes PSa and 3 a) are shown in FIG. 5.

[0084] 1.4 List of Oligonucleotides

[0085] In general, in the oligonucleotide sequences disclosed in Example1 and the successive examples below: lower case letters denote the siteof the DF508 mutation; promoter portions are shown underlined; portionsof probes used for detection purposes are indicated by italics; andcapture portions are shown in bold face. The 3′ phosphate groups on PSprobes (where included) are optional. Target oligonucleotide (Normalwild type CFTR DNA)5′ TTATGCCTGGCACCATTAAAGAAAATATCATCtttGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAAAGC3′ (Seq. ID No.16) CS probe (T7 promoter)5′ ATAGGAAACACCAAAGATGATATTTTCTTTAATACGACTCACTATA 3′ (Seq. ID No.17) PSprobe (T7 promoter with 3′ATT 5′ start sequence in target, and templateportion) 5′ CCTTGTCTCCGTTCT GGATATCACCCGATGTGTCTCCCTATAGTGAGTCGTA 3′(Seq. ID No.18) PSa probe (T7 promoter with 3′ATT 5′ start sequence intarget, and template portion with capture tail extended to 20 bases formore sensitive capture and detection, using probe 3a)5′ TGCCTCCTTGTCTCCGTTCT GGATATCACCCGATGTGTCTCCCTATAGTGAGTCGTA phosphate3′ (Seq. ID No.19) Probe 3 (with 5′ biotin to allow capture onstreptavidin coated plates) 5′ TGCCTCCTTGTCTCCGTTCT 3′ (Seq. ID No.20)Probe 3a (version of probe 3a extended by 5 bases to allow moresensitive capture of transcript from probe PSa)5′ TCCGCTGCCTCCTTGTCTCCGTTCT 3′ (Seq. ID No.21) Probe 4(europium-labelled) 5′ GGATATCACCCG 3′ (Seq. ID No.22)

Example 2 Transcription from a Split T3 RNA Polymerase Promoter at a 2½Way Junction at DF508

[0086] The example is illustrated schematically in FIG. 6. The completeT3 promoter is located towards the 3′ end of the CS probe (2). The firstthree bases of the T3 RNA polymerase promoter (5′AAA3′) in the CS probe(2) anneals to the 3′TTT5′ DF508 site (8) in the wild type target (6),and therefore the DF508 mutation will result in loss of the splitpromoter start, with subsequent loss of transcription. Hybridisation ofa PS probe (4), (at the 3′ end of which is the complement to the T3promoter minus three bases) to CS probe (2) forms a double strandedpromoter, made complete by the three bases (3′TTT 5′) in the target, andtherefore a split promoter is formed to yield a de novo synthesised RNA(14) in the presence of T3 RNA polymerase.

[0087] 2.1 Preparation of Oligonucleotides

[0088] The target oligonucleotides and probes are synthesised andpurified as described in Example 1.

[0089] 2.2 Split promoter probe and RNA synthesis

[0090] Hybridisation reactions comprise mixtures of DNA including targetoligonucleotide, PS and CS probes, together with relevant controlscomprising mixtures with and without target/probes PS and CS.Hybridisation reactions are established as described in Example 1.2, butusing the probe sequences detailed below and T3 RNA polymerase/buffer(Promega). The hybridisation mixture is then treated as described inExample 1, but using probe 3 and probe 4 sequences detailed below.

[0091] 2.3 Detection of RNA by Time Resolved Fluorescence (TRF)

[0092] 5 μl of reaction sample is added to the reaction mix consistingof 145 pi of Wallac assay buffer, 0.9 pmol of probe 3 and 0.3 pmol ofprobe 4 in a well of a Labsystems streptavidin coated microtitre plate,which is incubated at room temperature for 60 minutes. The assay is thenperformed as desribed above (at Example 1.3).

[0093] 2.4 List of Oligonucleotides Target oligonucleotide (Normal wildtype DNA)5′ TTATGCCTGGCACCATTAAAGAAAATATCATCtttGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAAAGC3′ (Seq. ID No.16) CS Probe (T3 promoter)5′ CTGTATCTATATTCATCATAGGAAACACCAAATTAACCCTCACTAAA 3′ (Seq. ID No.23) PSProbe (T3 promoter with 3′ TTT 5′ start sequence in target, and templateportion) 5′ CCTTGTCTCCGTTCTGGATATCACCCGATGTGATTCCCTTTAGTGAGGGTTAA phosphate 3′ (Seq. ID No.24)Probe 3 (with 5′ biotin to allow capture on streptavidin coated plates)5′ TGCCTCCTTGTCTCCGTTCT 3′ (Seq. ID No.20) Probe 4 (europium-labelled)5′ GGATATCACCCG 3′ (Seq. ID. No.22)

Example 3 Transcription from a Split T3 RNA Polymerase Promoter at a 2½Way Junction

[0094] The example is illustrated schematically in FIG. 7. The completeT3 promoter (the first three bases of which is 5′TTA 3′, a differentversion to that in example 2) is located towards the 3′ end of the CSprobe (2). The first three (5′) bases of the promoter sequence iscomplemented by three bases (3′AAT 5′), (8) in the target (6) (Target:Hepatitis B (Hep B) DNA). Hybridisation of a PS probe (4), (at the 3′end of which is the complement to the T3 promoter minus three bases) toCS probe (2) forms a double stranded promoter, made complete by thethree bases (3′AAT 5′) in the target (6), and therefore a split promoteris formed to yield a de novo synthesised RNA in the presence of T3 RNApolymerase.

[0095] 3.1 Preparation of Oligonucleotides

[0096] The target oligonucleotides and probes were synthesised andpurified as described in Example 1.

[0097] 3.2 Split Promoter Probe and RNA Synthesis

[0098] Hybridisation reactions are established as described in Example2.2 and treated as described in Example 1.2 above, but using the nucleicacid sequences detailed below. The resulting product is detected by thehybridisation of molecular beacon (see below).

[0099] 3.3 Detection of RNA by Molecular Beacon Assay

[0100] 5 μl of reaction sample is added to the reaction mix consistingof 145 μl of hybridisation solution and 2 pmol molecular beacon(fluorophore =FAM; quencher =methyl red), in a Labsystems WhiteMicrostrip microtitre plate, which is incubated in the dark at roomtemperature for 60 minutes. Fluorescence signal from the hybridisedbeacon/target is measured using the Wallac Victor 1420 MultilabelCounter, using the fluorescein protocol.

[0101] 3.4 List of Oligonucleotides Target (Hep B DNA)5′ GAGGCATAGCAGCAGGATGAAGAGGAAGATGATAAAACGCCGCAGACACA (Seq. ID No.25)TCCAGCGATAACCAGGACAGGTTGGAGGACAGGA 3′ CS Probe (T3 promoter)5′ TGGTTATCGCTGGATGTGTCTGCGGCGTTTTATTAACCCTCACTAAA 3′ (Seq. ID No.26) PSProbe (T3 promoter with 3′ AAT 5′ start sequence in target, and templateportion) molecular beacon sequence5′ GTTCTATCCTGCACCGCCGGAGCTTTCCACCCCTTCCCTTTAGTGAGGGTTA (Seq. ID No.27)A phosphate 3′ Molecular Beacon Oligonucleotide probe (comprising asequence derived from Streptomyces thermoalkatolerans, withcomplementary 5′ and 3′ ends) 5′ CGCGATCCTGCACCGCCGGAGCTTTCCACCCCGCG 3′(Seq. ID No.28)

Example 4 Transcription from a Split SP6 Promoter at a 2½ Way Junction

[0102] In this example, the target is wild type human DNA CFTR gene inwhich a deletion of TTT causes a cystic fibrosis-encoding mutationDF508.

[0103] The example is illustrated schematically in FIG. 8. The completeSP6 promoter is located towards the 3′ end of the CS probe (2). Thefirst three (5′) bases of the promoter sequence is complemented by threebases (3′TAA 5′) (8) in the target (6), and the CS probe (2) hybridisesto the target (6) in such a way that the 3′TTT 5′ in the wild type is 6bases downstream from the start of the promoter. Hybridisation of a PSprobe (4), (at the 3′ end of which is the complement to the SP6 promoterminus three bases) to CS probe (2) forms a double stranded promoter,made complete by the three bases (3′TAA 5′) in the target, and thereforea split promoter is formed to yield a de novo synthesised RNA (14) inthe presence of SP6 RNA polymerase.

[0104] 4.1 Preparation of Oligonucleotides

[0105] The target oligonucleotides and probes are synthesised andpurified as described in Example 1.

[0106] 4.2 Split promoter probe and RNA Synthesis

[0107] Hybridisation reactions are established and treated as describedin Example 1.2, but using the nucleic acid sequences detailed below andusing SP6 RNA polymerase/buffer (Promega).

[0108] 4.3 Detection of RNA by Time Resolved Fluorescence (TRF)

[0109] 5μl of reaction sample is added to the reaction mix consisting of145 μl of Wallac assay buffer, 0.9 pmol of probe 3 and 0.3 pmol of probe4 in a well of a Labsystems streptavidin coated microtitre plate, whichis incubated at room temperature for 60 minutes. The assay is thenperformed as described above (at section 1.3).

[0110] 4.4 List of Oligonucleotides Target oligonucleotide (Normal wildtype DNA)5′ TTATGCCTGGCACCATTAAAGAAAATATCATCtttGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAAAGC3′ (Seq. ID No.16) CS Probe (SP6 promoter)5′ ATTCATCATAGGAAACACCAAAGATGATATTTAGGTGACACTATA 3′ (Seq. ID No.29) PSProbe (SP6 promoter with 3′TAA5′ start sequence in target, and templateportion) 5′ CCTTGTCTCCGTTCTGGATATCACCCGATGTGGTATTCTATAGTGTCACCTA phosphate 3′ (Seq. ID No.30) Probe3 (with 5′ biotin to allow capture on streptavidin coated plates)5′ TGCCTCCTTGTCTCCGTTCT 3′ (Seq. ID No.20) Probe 4 (europium-labelled)5′ GGATATCACCCG 3′ (Seq. ID No.31)

Example 5 Transcription from a Split SP6 RNA Polymerase Promoter at a 2½Way Junction at DF508

[0111] The example is illustrated schematically in FIGS. 9A and 9B. Oneconformation of the DF508 CFTR mutation results in the loss of a 3′GAA5′ from the sequence 3′TAGAAA 5′, resulting in the creation of a 3′TAA5′ triplet (8) and thus an SP6 promoter start sequence (FIG. 9B). Hencea functional SP6 promoter is created using CS probe (2) and PS probe (4)with CFTR mutant DNA target (6), whereas no functional promoter iscreated using PS and CS probes with normal wild type DNA target (FIG.9A). Hybridisation of a PS probe (4), (at the 3′ end of which is thecomplement to the SP6 promoter minus three bases) to CS probe (2) formsa double stranded promoter, made complete by the three bases (3′TAA 5′)in the mutant target (6), and therefore a split promoter is formed toyield a de novo synthesised RNA in the presence of SP6 RNA polymerase.

[0112] 5.1 Preparation of Oligonucleotides

[0113] The target oligonucleotides and probes are synthesised andpurified as desribed in Example 1.

[0114] 5.2 Split promoter probe and RNA synthesis

[0115] Hybridisation reactions are established and treated as describedin Example 1.2, but using the nucleic acid sequences detailed below andusing SP6 RNA polymerase/buffer (Promega).

[0116] 5.3 Detection of RNA by Time Resolved Fluorescence (TRF)

[0117] 5μl of reaction sample is added to the reaction mix consisting of145 μl of Wallac assay buffer, 0.9 pmol of probe 3 and 0.3 pmol of probe4 in a well of a Labsystems streptavidin coated microtitre plate, whichis incubated at room temperature for 60 minutes. The assay is thenperformed as described above (section 1.3).

[0118] 5.4 List of Oligonucleotides Target Oligonucleotide (Normal wildtype DNA, no DF508 deletion)5′ GTTGGCATGCTTTGATGACGCTTCTGTATCTATATTCATCATAGGAAACACC (Seq. ID No.32)AaagATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGAAAACTGAGAACAG AATGAAATTCTTC 3′Target oligonucleotide (CF mutant DNA, with the DF508 deletion)5′ GTTGGCATGCTTTGATGACGCTTCTGTATCTATATTCATCATAGGAAACACCa (Seq. ID No.33)atGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGAAAACTGAGAACAGAATG AAATTCTTC 3′ CSProbe (SP6 promoter)5′ TTATGCCTGGCACCATTAAAGAAAATATCATTTAGGTGACACTATA 3′ (Seq. ID No.34) PSProbe (SP6 promoter with 3′ TAA 5′ start sequence formed by a DF508mutation in CFTR mutant DNA, and template portion) 5′ CCTTGTCTCCGTTCTGGATATCACCCGATGTGGTATTCTATAGTGTCACCT (Seq. ID No.30) A phosphate 3′Probe 3 (with 5′ biotin to allow capture on streptavidin coated plates)5′ TGCCTCCTTGTCTCCGTTCT 3′ (Seq. ID No.20) Probe 4 (europium-labelled)5′ GGATATCACCCG 3′ (Seq. ID No.22)

Example 6 Transcription from a Split T7 RNA Polymerase Promoter at a 3Way Junction

[0119] In this example, the target is wild type human DNA CFTR gene, atwhich a deletion of TTT causes a cystic fibrosis-encoding mutationDF508.

[0120] The complete T7 promoter is located towards the 3′ end of a CSprobe. The first three (5′) bases of this sequence complement threebases in the target. Hybridisation of a PS probe, (which has thecomplement to the T7 promoter minus three bases, and a complement to thetarget DNA) to CS probe and the target forms a double stranded promoter,made complete by the three bases in the target, and therefore a splitpromoter is formed to yield a de novo synthesised RNA in the presence ofT7 RNA polymerase.

[0121] 6.1 Preparation of Oligonucleotides

[0122] The target oligonucleotide and probes were synthesised andpurified as described in Example 1.

[0123] 6.2 Split Promoter Probe and RNA Synthesis

[0124] Hybridisation reactions were established and treated as describedin Example 1.2, but using the nucleic acid sequences detailed below. Theresulting product was immobilised by hybridisation to a specificbiotinylated oligonucleotide (probe 3) which was in turn bound to astreptavidin coated well of a microtitre plate. The immobilised productwas detected by colorimetry via the hybridisation of probe 4, analkaline phosphatase-labelled oligonucleotide probe (see below).

[0125] 6.3 Detection of RNA by Colorimetry

[0126] 5μl of reaction sample was added to the reaction mix consistingof 145 μl hybridisation buffer (20 mM EDTA pH 8.0,1 M NaCl, 50 mM Tris,0.1% bovine serum albumin, mixture adjusted to pH 8.0 with HCl), 0.9pmol of probe 3 and 12.7 pmol of 5 probe 4 in a well of a Labsystemsstreptavidin coated microtite plate, which was incubated at roomtemperature for 60 minutes. Unbound material was removed by washing thewells 4× with 200 μl of wash solution (0.25 M Tris, 0.69 M NaCl, 13.4 mMKCl, adjusted to pH 8.0 with HCl), and 1× with substrate buffer (used asa 1× solution, made from a 5× concentrate stock obtained from BoehringerMannheim 726915). 180 μl of substrate buffer containing 5 mg/ml of4-nitrophenyl phosphate was added the well, and colour development wasmeasured by optical density at 405 nm using a Labsystems integrated EIAManagement system plate reader, readings taken every 2 minutes for 30minutes. The results obtained are shown in FIG. 10.

[0127] 6.4 List of Oligonucleotides Target oligonucleotide (Normal wildtype DNA) 5′ GTTGGCATGCTTTGATGACGCTTCTGTATCTATATTCATCATAGGAAACACC (Seq.ID No.32) aaaGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGAAAACTGAGAACAGAATGAAATTCTTC 3′ CS Probe (T7 promoter)5′ CAGTTTTCCTGGATTATGCCTGGCACCATTAATACGACTCACTATA 3′ (Seq. ID No.35) PSProbe (T7 promoter with 3′ ATT 5′ start sequence in target, and templateportion) 5′ CCTTGTCTCCGTTCT GGATATCACCCGATGTGTCTCCCTATAGTGAGTCGTA (Seq.ID No.36) AGAAAATATCATCTTTGGTGTTTCCTATGATG 3′ Probe 3 (with 5′ biotin toallow capture on streptavidin coated plates) 5′ TGCCTCCTTGTCTCCGTTCT 3′(Seq. ID No.20) Probe 4 (alkaline phosphatase-labelled)5′ GGATATCACCCGATGTG 3′ (Seq. ID No.37)

Example 7 Transcription from a Split T7 RNA Polymerase Promoter, andAmplification without the use of DNA Polymerase Extension

[0128] This example demonstrates the creation of a functional DNAdependent RNA polymerase promoter as a result of the formation of anucleic acid complex comprising target nucleic acid (Target: wild typehuman DNA: cystic fibrosis transmembrane conductance regulator gene(CFTR) at which a deletion of TTT causes a cystic fibrosis-encodingmutation DF508), a partly complementary oligonucleotide and a promoterstrand probe.

[0129] The example is illustrated schematically in FIG. 12. The completeT7 promoter is located towards the 3′ end of a CS probe (2). The firstthree (5′) bases of the promoter sequence is complemented by three bases(3′ATT 5′) (8) in target (6), and CS probe (2) hybridises to target (6)in such a way that the 3′TTT 5′ in the wild type sequence is 14 basesdownstream from the start of the promoter. Hybridisation of a PS probe(4), (at the 3′ end of which is the complement to the T7 promoter minusthree bases) to CS probe (2) forms a double stranded promoter, madecomplete by the three bases (8) in target (6), and therefore a splitpromoter is formed to yield a de novo synthesised RNA (14) in thepresence of T7 RNA polymerase.

[0130] The de novo synthesised RNA species is then amplified, asrepresented in FIG. 13. Referring to FIG. 13, RNA molecule (14) containsan overlap sequence, a second promoter sequence (SP6, designated as Pr(2) in FIGS. 12 and 13) and a further 6 bases to compensate for possibleearly termination of transcription. This molecule (14) anneals to addedDNA probe 3 (50 in FIG. 13) in the amplification scheme, creating adouble stranded SP6 promoter and thus initiates the amplification cycle(step i). The RNA transcript (52) from probe 3 (50) includes a differentoverlap sequence to that of RNA molecule (14), a sequence for T3 RNApolymerase promoter (promoter 3 or Pr 3) and a further 6 bases. Thismolecule (52) anneals (step ii) to added DNA probe 4, (54 in FIG. 13)creating a double stranded T3 promoter which initiates the transcription(step iii) of RNA (56) anneals (step iv) to probe 3 (50) in acontinuation of the amplification cycle. Note that promoters 1, 2 and 3need not be necessarily T7, SP6 and T3 RNA polymerase promotersrespectively: they could be used in a different order to that shown, orone or more other RNA promoters not discussed here may alternatively beemployed.

[0131] In contrast to the amplification system illustrated in FIG. 3, anactive RNA promoter is formed directly by hybridisation of appropriatenucleic acid sequences in the system described above, there is noextension required for promoter formation.

[0132] 7.1 Preparation of Oligonucleotides

[0133] The target oligonucleotides and probes are synthesised andpurified as described in Example 1.

[0134] 7.2 RNA Synthesis by Split Promoter and Amplification Cycle

[0135] Hybridisation reactions comprise mixtures of DNA including targetoligonucleotide, PS and CS probes, probe 3 and probe 4 together withrelevant controls comprising mixtures with and without target/PS or CSprobes and probes 3 and 4. For hybridisation reactions, 40 fmol oftarget oligonucleotide is mixed with 40 fmol each of probes PS, CS, 3and 4 in a solution containing 4 μl 5× RNA polymerase buffer (giving 1×concentrations of 40 mM Tris (pH7.9), 6 mM MgCl₂, 2 mM spermidine and 10mM NaCl) and distilled water to a final volume of 20 pi (following finaladdition of T7, SP6 and T3 RNA polymerases and rNTP mix). The mixture isheated to 90° C. for 3 minutes to denature the nucleic acids, incubatedon ice for 2 minutes, and equilibrated to 37° C. for 1 minute. Probesare annealed and transcribed at 37° C. for 180 minutes by addition of 40units of each RNA polymerase (Promega) and 120 nmoles of each rNTP(Pharmacia Biotech). DNA oligonucleotides are removed from the reactionmix by heating to 90° C. for 3 minutes and incubating on ice for 2minutes followed by the addition of 4 units of DNase I (Ambion) andincubating at 37° C. for 20 minutes prior to end detection. One (orpotentially both) of the resulting products (RNAs 14&52) may be detectedby the hybridisation of molecular beacon (see below).

[0136] 7.3 Detection of RNA by Molecular Beacon Assay

[0137] 5 μl of reaction sample was added to the reaction mix consistingof 145 μl of hybridisation solution and 2 pmol molecular beacon (5′fluorophore=FAM; 3′ quencher=methyl red probe 5), in a Labsystems WhiteMicrostrip well plate, which is incubated in the dark at roomtemperature for 60 minutes. Fluorescence signal from the hybridisedbeacon/target is measured using the Wallac Victor 1420 MultilabelCounter, using the fluorescein protocol.

[0138] 7.4 List of Oligonucleotides Target oligonucleotide (Normal wildtype CFTR DNA) 5′ TTATGCCTGGCACCATTAAAGAAAATATCATCtttGGTGTTTCCTATGATGA(Seq. ID No. 16) ATATAGATACAGAAGCGTCATCAAAGC 3′ CS probe (T7 promoter)5 ′ATAGGAAACACCAAAGATGATATTTTCTTTAATACGACTCACTATA 3′ (Seq. ID No. 17) PSProbe (T7 promoter (Pr1) with 3′ ATT 5′ start sequence in target, andtemplate portion which encodes transcript with SF6 promoter (Pr2))            SP6 promoter                   T7 promoter5′ GTATTCTATAGTGTCACCTAAATATTTCACGCGATAAGTATCTCCCTATAGTGAGTCGTA 3′ (Seq.ID No. 38) Probe 3 (first DNA oligo in amplification cycle with SP6promoter (Pr2), encoding transcript with T3 promoter (Pr3))            T3 promoter                    molecular5′ CTTCCCTTTAGTGAGGGTTAATAATGCCTCCTTGTCTCCGTTCTCGTGGAAT (Seq. ID No. 39)beacon sequence                SP6 promoterGTTGCCCACACCTAGTGCCCACGTATTCTATAGTGTCACCTAAATATTTCACGCGAT 3′ Probe 4(second DNA oligo in amplification cycle with T3 promoter (Pr3) encodingtranscript with SP6 promoter (Pr2))             SP6promoter                molecular5′ GTATTCTATAGTGTCACCTAAATATTTCACGCGATAAGTACGTGGAATGTTG (Seq. ID No. 40)beacon sequence           T3 promoterCCCACACCTAGTGCCCACCTTCCCTTTAGTGAGGGTTAATAATGCCTCCTTGTGTCC 3′ Probe 5(molecular beacon: 5′ fluorescent label and 3′ quencher) FAM5′ CGCGCGTGGAATGTTGCCCACACCTAGTGCCCACCGCG 3′ Methyl red (Seq. ID No. 41)

Example 8 Transcription from a Split T7 RNA Pol Promoter 2½ WayJunction, using an RNA Target

[0139] This example involves the use of an RNA target (based on the CFTRsequence). The complete T7 promoter is located towards the 3′ end of CSprobe. The first three (5′) bases of the promoter sequence iscomplemented by three bases (3′AUU 5′) in the target, when CS probehybridises to the target. Hybridisation of a second oligonucleotide (PSprobe, at the 3′ end of which is the complement to the T7promoter minusthree bases) to CS probe forms a double stranded promoter, made completeby the three bases in the target, and therefore a split promoter isformed to yield a de novo synthesised RNA in the presence of T7 RNApolymerase. This reaction was compared to a control reaction which useda DNA version of the CFTR target. This example shows that sample RNAcould be used as a target for the split promoter (i.e. that thepolymerase will recognise a promoter which comprises at least threebases of RNA rather than DNA). Furthermore, the resulting RNA transcriptcould be further amplified using a second split promoter. The RNA signalfrom this second promoter could be again amplified by re-forming theprevious split promoter, and so on, in an amplification cycle relying onthe presence of T7 RNA polymerase and rNTPs only (e.g. as illustratedschematically in FIG. 15).

[0140] 8.1 Preparation of Oligonucleotides

[0141] The target oligonucleotides and probes were synthesised andpurified as described in example 1.

[0142] 8.2a Synthesis and Quantification of RNA Target

[0143] RNA target molecules were prepared by transcription with T7 RNApolymerase, under standard conditions, of a double stranded DNAoligonucleotide prepared so as to include a T7 polymerase promoter. DNAoligonucleotides were then removed from the reaction mix by addition of3 units of DNase I (Ambion) and incubation at 37° C. for 10 minutes,followed by heat inactivation of the enzyme at 90° C. for 3minutes. Thetranscript was quantified using the RiboGreen RNA Quantitation Kit(Molecular Probes, R-11490).

[0144] For quantification, 5 μl of the reaction mixes, or of dilutionsin TE were added to 95 μl of TE in the well of a Labsystems WhiteMicrostrip well plate, together with 100 μl of the Quantitation Reagent(a 1/2000 dilution of the Quantitation Reagent in TE, according to themanufacturer's instructions). The plate was shaken at 200 rpm for 5minutes at 22° C., followed by detection of fluorescence signal from theintercalated fluorophore/RNA measured using the Wallac Victor 1420Multilabel Counter, using the fluorescein protocol. The fluoresencesignal value was converted to pmol RNA by comparison to a standard curvemeasured in the same way as the transcript, but using a standardsynthetic RNA (probe 3, below). The quantified RNA was stored in 10 μlaliquots at −80° C.

[0145] 8.2b Split Promoter Probe and RNA Synthesis

[0146] Hybridisation reactions comprised mixtures of DNA includingtarget RNA or DNA oligonucleotide, CS probe and PS probe together withrelevant controls comprising mixtures with and without target/probes CSand PS. For hybridisation reactions, 50 fmol of target RNA or DNAoligonucleotide was mixed with 50 fmol of CS probe and 50 fmol of PSprobe in a solution containing 28.3 pi T7 RNA polymerase buffer (giving1× concentrations of 40 mM Tris (pH8.1), 20 mM MgCl₂, 1 mM spermidine, 5mM DTT (Promega P117C), 80 mg/ml PEG 8000, 50 μg/ml BSA, and 0.01%Triton X-100: Milligan et al., 1987, Nucleic Acids Research, volume 15,pp. 8783-8798) and distilled water to a final volume of 50 pi (followingfinal addition of T7 RNA polymerase and rNTP mix). The mixture washeated to 90° C. for 3 minutes to denature the nucleic acids, thencooled to 10° C. at 0.1° C. per second for hybridisation. Probes wereannealed and transcribed at 37° C. for 180 minutes by addition of 25units of T7 RNA polymerase and 40 nmoles of each rNTP. DNAoligonucleotides were removed from the reaction mix by the addition of 3units of DNase I and incubating at 37° C. for 10 minutes, followed byheating to 90° C. for 3 minutes, and cooling to 15° C., prior to enddetection. The resulting product was immobilised by hybridisation to aspecific biotinylated oligonucleotide (probe 4, below) which was in turnbound to a streptavidin coated well. The immobilised product wasdetected by colorimetry via the hybridisation of probe 5 (see below), analkaline phosphatase-labelled oligonucleotide probe.

[0147] 8.3 Detection of RNA by Colorimetry

[0148] 5 μl of reaction sample or dilutions was added to the reactionmix consisting of 145 μl hybridisation buffer (20 mM EDTA pH 8.0, 1 MNaCl, 50 mM Tris, 0.1% bovine serum albumin, mixture adjusted to pH 8.0with HCl), 0.9 pmol of probe 4 and 6 pmol of probe 5 in a Labsystemsstreptavidin coated well plate, which was incubated at 22° C. for 60minutes. Unbound material was removed by washing the wells 4× with 200μl of wash solution (0.25 M Tris, 0.69 M NaCl, 13.4 mM KCl, adjusted topH 8.0 with HCl), and 1× with substrate buffer (used as a 1× solution,made from a 5× concentrate stock obtained from Boehringer Mannheim). 180μl of substrate buffer containing 5 mg/ml of 4-nitrophenyl phosphate wasadded to the well, and colour development was measured by opticaldensity at 405 nm using a Labsystems integrated EIA Management systemplate reader, readings taken every 2 minutes for 30 minutes. Results areshown in FIG. 14.

[0149]FIG. 14 is a bar chart showing the amount of RNA produced (inpicomoles) using either an RNA target (left hand columns) or a DNAtarget (right hand columns). For each group of columns, (1) gives theresults obtained for mixtures comprising target and first and secondprobes; (2) gives the results obtained in the absence of target; and (3)gives the results obtained in the absence of target and first probe.

[0150] It can be seen that, although a DNA target results in theproduction of more RNA, both DNA and RNA targets can be usedsuccessfully to form functional “split” promoters. In either case thebackground signal is very low.

[0151] 8.4 List of Oligonucleotides RNA target (sequence based on normalwild type CFTR DNA)5′GGGAGAUGAUGACGCUUCUGUAUCUAUAUUCAUCAUAGGAAACACCAAAGAUGAUAUUUUCUUUAAUGGUGCCAGGCA(Seq. ID No. 42) UAAUCCAGGAAAACUGAGAACA3′ DNA target oligonucleotide(Normal wild type CFTR DNA)5′GTTGGCATGCTTTGATGACGCTTCTGTATCTATATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGC(Seq. ID No. 32) CAGGCATAATCCAGGAAAACTGAGAACAGAATGAAATTCTTC3′ CS probe(T7 promoter with complementary 3′ATT5′ start sequence in target)5′CAGTTTTCCTGGATTATGCCTGGCACCATTAATACGACTCACTATA3′ (Seq. ID No. 35) PSprobe (T7 promoter and template) 5′TGCCTCCTTGTCTCCGTTCTGGATATCACCCGATGTGTCTCCCTATAGTGAGTCGTA3′ (Seq. ID No. 19) Probe 3 (RNAcontrol for constructing the standard curve in the RiboGreen assay)5′GGGAGACACAUCGGGUGAUAUCCAGAACGGAGACAAGG3′ (Seq. ID No. 43) Probe 4(with 5′ biotin to allow capture on streptavidin coated plates)5′TCCGCTGCCTCCTTGTCTCCGTTCT3′ (Seq. ID No. 21) Probe 5 (alkalinephosphatase-labelled) 5′GGATATCACCCG3′ (Seq. ID No. 22)

Example 9 Transcription of a Ribozyme from a Split T7 RNA Pol promoterat a 2½ Way Junction

[0152] In this example RNA produced from a split promoter has thesequence of a known ribozyme (Clouet-D'Orval & Uhlenbeck, 1996 RNA 2(5):483-491) and can bind to a dual labelled single stranded oligonucleotideto form a functional ribozyme. Cleavage of the labelled oligonucleotideat a specific site will then generate a signal. The complete T7 promoteris located towards the 3′ end of CS probe. The first three (5′) bases ofthe promoter sequence is complemented by three bases (3′ATT 5′) in thetarget, when CS probe hybridises to the target. Hybridisation of asecond oligonucleotide (PS probe, at the 3′ end of which is thecomplement to the T7 promoter minus three bases) to CS probe forms adouble stranded promoter, made complete by the three bases in thetarget, and therefore a split promoter is formed to yield a de novosynthesised RNA ribozyme in the presence of T7 RNA polymerase.

[0153] 9.1 Preparation of Oligonucleotides

[0154] The target oligonucleotides and probes are synthesised andpurified as described in example 1.

[0155] 9.2 Split Promoter Probe and RNA Synthesis

[0156] Hybridisation reactions comprise mixtures of DNA including targetoligonucleotide, CS probe and PS probe together with relevant controlscomprising mixtures with and without target/probes CS and PS. Forhybridisation reactions, 40 fmol of target oligonucleotide is mixed with40 fmol of CS probe and 40 fmol of PS probe in a solution containing 4μl 5× T7 RNA polymerase buffer (giving 1× concentrations of 40 mM Tris(pH7.9), 6 mM MgCl₂, 2 mM spermidine and 10 mM NaCl) and distilled waterto a final volume of 20 μl (following final addition of T7 RNApolymerase and rNTP mix). The mixture is heated to 90° C. for 3 minutesto denature the nucleic acids, then cooled to 10° C. at 0.1° C. persecond for hybridisation. Probes are annealed and transcribed at 37° C.for 180 minutes by addition of 40 units of T7 RNA polymerase and 40nmoles of each rNTP. DNA oligonucleotides are removed from the reactionmix by the addition of 3 units of DNase I and incubating at 37° C. for20 minutes prior to end detection.

[0157] 9.3 Detection of Synthesised RNA

[0158] 5 μl aliquots of sample or 5 μl of a suitable dilution of thetreated assay sample are added to 100 μl buffer (50 mM Tris-HCl pH7.5,20 mM MgCl₂, 10% ethanol), followed by 10 pmol probe 3. Thisdouble-labelled RNA (5′-Tamra, 3′-Fam) is the ribozyme substrate. TheRNA product of the 2½ way junction (formed in the presence of specifictarget) is designed to be the corresponding “hammerhead” ribozyme. Probe3 therefore anneals to the RNA product, creating a functional ribozyme.Ribozyme cleavage of the substrate, which results in the removal of thequencher from the fluorophore, can be monitored by FluorescenceResonance Energy Transfer (Tamra excitation at 546 nm, emission at 579nm). Alternatively, substrate cleavage could be measured by a decreasein fluorescence polarisation. Since substrate turnover is possible, alevel of amplification may be achieved during the detection process.

[0159] Alternative Real Time Detection System

[0160] Real time detection would be possible if the ribozyme substratemolecule is included in the extension/transcription reaction mixture,under suitable buffer conditions.

[0161] Alternative Detection Systems:

[0162] The RNA product could include a capture sequence, allowing it tobe captured on to a streptavidin-coated well via a biotinylated captureprobe. After wash steps to remove unbound material, probe 3 could beadded and ribozyme cleavage could be monitored as described above.

[0163] Alternative labels could be attached to the ribozyme substratemolecule.

[0164] 9.4 List of Oligonucleotides Target oligonucleotide (Normal wildtype CFTR DNA)5′GTTGGCATGCTTTGATGACGCTTCTGTATCTATATTCATCATAGGAAACACCaaaGATGAT (Seq. IDNo. 32) ATTTTCTTTAATGGTGCCAGGCATAATCCAGGAAAACTGAGAACAGAATGAAATTCTTC3′ CSprobe (T7 promoter with 3′ATT5′ start sequence in target)5′CAGTTTTCCTGGATTATGCCTGGCACCATTAATACGACTCACTATA3′ (Seq. ID No. 35) PSprobe (T7 promoter and template, which encodes the ribozyme)5′GAATCTCATCAGTAGCGAGTTCTCTCTCCCTATAGTGAGTCGTA3′ (Seq. ID No. 44) Probe3 (ribozyme substrate) 5′Tamra-GAAUCGAAACGCGAAAGCGUCUAGCGU-FAM3′ (Seq.ID No. 45)

Example 10

[0165] An experiment was conducted to determine the optimum sequence of+12 region for most efficient transcription by T7 RNA polymerase.

[0166] Accordingly a series of second probe molecules were prepared,each comprising identical T7 promoter, detection and capture sequencesbut having different +12 sequences adjacent to the T7 promoter sequence.These probes were hybridised to a complementary 22 base oligonucleotide(containing the complementary strand of the T7 promoter) under identicalconditions, and the amount of RNA produced was determined as describedin the previous examples.

[0167] Table 2 below shows the relative “RNA transcription factor” foreach of the different +12 sequences tested. TABLE 2 Alternative templateT7 +1 to +12 sequences in descending order of transcription efficiency.+1 to +12 sequence RNA transcription factor 5′ GTTCTCTCTCCC 3′ 1425′ GCTCTCTCTCCC 3′ 115 5′ GTTGTGTCTCCC 3′ 110 5′ GATGTGTCTCCC 3′ 1055′ ATCCTCTCTCCC 3′ 96 5′ GTTCTCGTGCCC 3′ 84 5′ ATCCTCGTGCCC 3′ 765′ GCTCTCGTGCCC 3′ 64 5′ GTTGTGGTGCCC 3′ 21

[0168]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 45 <210> SEQ ID NO 1<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Bacteriophage T3 <220>FEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)..(18) <223> OTHERINFORMATION: 5′ TO 3′ <400> SEQUENCE: 1 aaattaaccc tcactaaa 18 <210> SEQID NO 2 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: BacteriophageT3 <220> FEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)..(18)<223> OTHER INFORMATION: 5′ TO 3′ <221> NAME/KEY: promoter <222>LOCATION: Complement ((1)..(18)) <400> SEQUENCE: 2 tttagtgagg gttaattt18 <210> SEQ ID NO 3 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Bacteriophage T7 <220> FEATURE: <221> NAME/KEY: promoter <222> LOCATION:(1)..(17) <223> OTHER INFORMATION: 5′ TO 3′ <400> SEQUENCE: 3 taatacgactcactata 17 <210> SEQ ID NO 4 <211> LENGTH: 17 <212> TYPE: DNA <213>ORGANISM: Bacteriophage T7 <220> FEATURE: <221> NAME/KEY: promoter <222>LOCATION: Complement ((1)..(17)) <400> SEQUENCE: 4 tatagtgagt cgtatta 17<210> SEQ ID NO 5 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:Bacteriophage SP6 <220> FEATURE: <221> NAME/KEY: promoter <222>LOCATION: (1)..(17) <223> OTHER INFORMATION: 5′ TO 3′ <400> SEQUENCE: 5atttaggtga cactata 17 <210> SEQ ID NO 6 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Bacteriophage SP6 <220> FEATURE: <221> NAME/KEY:promoter <222> LOCATION: (1)..(17) <223> OTHER INFORMATION: 3′ TO 5′<400> SEQUENCE: 6 tatagtgtca cctaaat 17 <210> SEQ ID NO 7 <211> LENGTH:12 <212> TYPE: DNA <213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 7gttctctctc cc 12 <210> SEQ ID NO 8 <211> LENGTH: 12 <212> TYPE: DNA<213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 8 gctctctctc cc 12<210> SEQ ID NO 9 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM:Bacteriophage T7 <400> SEQUENCE: 9 gttgtgtctc cc 12 <210> SEQ ID NO 10<211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Bacteriophage T7 <400>SEQUENCE: 10 gatgtgtctc cc 12 <210> SEQ ID NO 11 <211> LENGTH: 12 <212>TYPE: DNA <213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 11 atcctctctccc 12 <210> SEQ ID NO 12 <211> LENGTH: 12 <212> TYPE: DNA <213>ORGANISM: Bacteriophage T7 <400> SEQUENCE: 12 gttctcgtgc cc 12 <210> SEQID NO 13 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: BacteriophageT7 <400> SEQUENCE: 13 atcctcgtgc cc 12 <210> SEQ ID NO 14 <211> LENGTH:12 <212> TYPE: DNA <213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 14gctctcgtgc cc 12 <210> SEQ ID NO 15 <211> LENGTH: 12 <212> TYPE: DNA<213> ORGANISM: Bacteriophage T7 <400> SEQUENCE: 15 gttgtggtgc cc 12<210> SEQ ID NO 16 <211> LENGTH: 79 <212> TYPE: DNA <213> ORGANISM:Human <220> FEATURE: <221> NAME/KEY: gene <222> LOCATION: (1)..(79)<223> OTHER INFORMATION: Normal wild type CFTR oligonucleotide <400>SEQUENCE: 16 ttatgcctgg caccattaaa gaaaatatca tctttggtgt ttcctatgatgaatatagat 60 acagaagcgt catcaaagc 79 <210> SEQ ID NO 17 <211> LENGTH:46 <212> TYPE: DNA <213> ORGANISM: CFTR CS Probe with T7 Promoter <400>SEQUENCE: 17 ataggaaaca ccaaagatga tattttcttt aatacgactc actata 46 <210>SEQ ID NO 18 <211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: CFTR PSProbe with T7 Promoter <400> SEQUENCE: 18 ccttgtctcc gttctggatatcacccgatg tgtctcccta tagtgagtcg 50 <210> SEQ ID NO 19 <211> LENGTH: 57<212> TYPE: DNA <213> ORGANISM: PSa Probe with T7 Promoter <400>SEQUENCE: 19 tgcctccttg tctccgttct ggatatcacc cgatgtgtct ccctatagtgagtcgta 57 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Synthetic Oligonucleotide Probe <400> SEQUENCE: 20 tgcctccttgtctccgttct 20 <210> SEQ ID NO 21 <211> LENGTH: 25 <212> TYPE: DNA <213>ORGANISM: Synthetic Oligonucleotide Probe <400> SEQUENCE: 21 tccgctgcctccttgtctcc gttct 25 <210> SEQ ID NO 22 <211> LENGTH: 12 <212> TYPE: DNA<213> ORGANISM: Synthetic Oligonucleotide Probe <400> SEQUENCE: 22ggatatcacc cg 12 <210> SEQ ID NO 23 <211> LENGTH: 47 <212> TYPE: DNA<213> ORGANISM: CS Probe with T3 Promoter <400> SEQUENCE: 23 ctgtatctatattcatcata ggaaacacca aattaaccct cactaaa 47 <210> SEQ ID NO 24 <211>LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: PS Probe with T3 Promoter<400> SEQUENCE: 24 ccttgtctcc gttctggata tcacccgatg tgattccctttagtgagggt taa 53 <210> SEQ ID NO 25 <211> LENGTH: 84 <212> TYPE: DNA<213> ORGANISM: Hep B Oligonucleotide <400> SEQUENCE: 25 gaggcatagcagcaggtaga agaggaagat gataaaacgc cgcagacaca tccagcgata 60 accaggacaggttggaggac agga 84 <210> SEQ ID NO 26 <211> LENGTH: 47 <212> TYPE: DNA<213> ORGANISM: Hep B CS Probe with T3 Promoter <400> SEQUENCE: 26tggttatcgc tggatgtgtc tgcggcgttt tattaaccct cactaaa 47 <210> SEQ ID NO27 <211> LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: Hep B PS Probe withT3 Promoter <400> SEQUENCE: 27 gttctatcct gcaccgccgg agctttccaccccttccctt tagtgagggt taa 53 <210> SEQ ID NO 28 <211> LENGTH: 35 <212>TYPE: DNA <213> ORGANISM: Streptomyces thermoalkatoleransOligonucleotide Probe <400> SEQUENCE: 28 cgcgatcctg caccgccggagctttccacc ccgcg 35 <210> SEQ ID NO 29 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: CFTR CS Probe with SP6 Promoter <400> SEQUENCE: 29attcatcata ggaaacacca aagatgatat ttaggtgaca ctata 45 <210> SEQ ID NO 30<211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: CFTR PS Probe with SP6Promoter <400> SEQUENCE: 30 ccttgtctcc gttctggata tcacccgatg tggtattctatagtgtcacc ta 52 <210> SEQ ID NO 31 <211> LENGTH: 12 <212> TYPE: DNA<213> ORGANISM: Synthetic Oligonucleotide Probe <400> SEQUENCE: 31ggatatcacc cg 12 <210> SEQ ID NO 32 <211> LENGTH: 120 <212> TYPE: DNA<213> ORGANISM: Wild Type CFTR Oligonucleotide <400> SEQUENCE: 32gttggcatgc tttgatgacg cttctgtatc tatattcatc ataggaaaca ccaaagatga 60tattttcttt aatggtgcca ggcataatcc aggaaaactg agaacagaat gaaattcttc 120<210> SEQ ID NO 33 <211> LENGTH: 117 <212> TYPE: DNA <213> ORGANISM:Mutant CFTR Oligonucleotide <400> SEQUENCE: 33 gttggcatgc tttgatgacgcttctgtatc tatattcatc ataggaaaca ccaatgatat 60 tttctttaat ggtgccaggcataatccagg aaaactgaga acagaatgaa attcttc 117 <210> SEQ ID NO 34 <211>LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Mutant CFTR CS Probe with SP6Promoter <400> SEQUENCE: 34 ttatgcctgg caccattaaa gaaaatatca tttaggtgacactata 46 <210> SEQ ID NO 35 <211> LENGTH: 46 <212> TYPE: DNA <213>ORGANISM: Wild Type CFTR CS Probe with T7 Promoter <400> SEQUENCE: 35cagttttcct ggattatgcc tggcaccatt aatacgactc actata 46 <210> SEQ ID NO 36<211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Wild Type CFTR CS Probewith T7 Promoter <400> SEQUENCE: 36 ccttgtctcc gttctggata tcacccgatgtgtctcccta tagtgagtcg taagaaaata 60 tcatctttgg tgtttcctat gatg 84 <210>SEQ ID NO 37 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: SyntheticOligionucleotide Probe <400> SEQUENCE: 37 ggatatcacc cgatgtg 17 <210>SEQ ID NO 38 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Wild TypeCFTR PS Probe with T7 and SP6 Promoter Sequences <400> SEQUENCE: 38gtattctata gtgtcaccta aatatttcac gcgataagta tctccctata gtgagtcgta 60<210> SEQ ID NO 39 <211> LENGTH: 109 <212> TYPE: DNA <213> ORGANISM:CFTR Oligonucleotide Probe with T3 and SP6 Promoter Sequence <400>SEQUENCE: 39 cttcccttta gtgagggtta ataatgcctc cttgtctccg ttctcgtggaatgttgccca 60 cacctagtgc ccacgtattc tatagtgtca cctaaatatt tcacgcgat 109<210> SEQ ID NO 40 <211> LENGTH: 109 <212> TYPE: DNA <213> ORGANISM:CFTR Oligonucleotide Probe with SP6 and T3 Promoter Sequence <400>SEQUENCE: 40 gtattctata gtgtcaccta aatatttcac gcgataagta cgtggaatgttgcccacacc 60 tagtgcccac cttcccttta gtgagggtta ataatgcctc cttgtctcc 109<210> SEQ ID NO 41 <211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:Synthetic Probe <400> SEQUENCE: 41 cgcgcgtgga atgttgccca cacctagtgcccaccgcg 38 <210> SEQ ID NO 42 <211> LENGTH: 100 <212> TYPE: RNA <213>ORGANISM: CFTR Nucleotide Target <400> SEQUENCE: 42 gggagaugaugacgcuucug uaucuauauu caucauagga aacaccaaag augauauuuu 60 cuuuaauggugccaggcaua auccaggaaa acugagaaca 100 <210> SEQ ID NO 43 <211> LENGTH: 38<212> TYPE: RNA <213> ORGANISM: RNA Control Sequence <400> SEQUENCE: 43gggagacaca ucgggugaua uccagaacgg agacaagg 38 <210> SEQ ID NO 44 <211>LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Ribozyme PS Probe with T7Promoter <400> SEQUENCE: 44 gaatctcatc agtagcgagt tctctctccc tatagtgagtcgta 44 <210> SEQ ID NO 45 <211> LENGTH: 27 <212> TYPE: RNA <213>ORGANISM: Human Ribozyme Substrate Probe <400> SEQUENCE: 45 gaaucgaaacgcgaaagcgu cuagcgu 27

1. A method of detecting the presence of a nucleic acid target sequenceof interest, the method comprising the steps of: (a) adding first andsecond nucleic acid probes to a sample comprising the sequence ofinterest, so as to form a complex comprising three strands of nucleicacid, wherein the first probe comprises the full length sequence of afirst strand of a double stranded promoter, the target sequencecomprises an end part of a second strand of the double stranded promoterwhich is complementary to a part of the first strand, and the secondprobe comprises the rest of the second strand of the double strandedpromoter which is complementary to a part of the first strand, such thata functional promoter is formed when the first probe is hybridised toboth the target sequence and to the second probe; (b) adding apolymerase which recognises the promoter, so as to cause the de novosynthesis of nucleic acid from the promoter present in the complex; and(c) detecting directly or indirectly the de novo synthesised nucleicacid:
 2. A method according to claim 1, wherein the promoter is an RNApolymerase promoter and the de novo synthesised nucleic acid is RNA. 3.A method according to claim 1 or 2, wherein the promoter is recognisedby T3, T7or SP6 RNA polymerase or a mutant form thereof.
 4. A methodaccording to any one of the preceding claims, wherein the second probecomprises a template portion which may act as a template for synthesisof nucleic acid from the functional promoter.
 5. A method according toclaim 4, wherein the second probe comprises a +12 region adjacent to thepromoter to optimise transcription from the promoter.
 6. A methodaccording to claim 5, wherein the second probe comprises a +12 regionsequence selected from the group consisting of:(5′® 3′)   GTTCTCTCTCCC;    GCTCTCTCTCCC;    GTTGTGTCTCCC;   GATGTGTCTCCC; ATCCTCTCTCCC; GTTCTCGTGCCC; ATCCTCGTGCCC;   GCTCTCGTGCCC; and GTTGTGGTGCCC.


7. A method according to any one of claims 4, 5 or 6, wherein thetemplate portion, when copied by the polymerase, provides a sequencewhich can act as an RNA polymerase promoter, or may be used fordetection and/or capture at a solid surface.
 8. A method according toany one of claims 4-7, wherein the template portion, when copied by thepolymerase, provides a sequence which hybridises with a molecularbeacon.
 9. A method according to any one of claims 4-7, wherein thetemplate portion, when copied by the polymerase, provides a sequencewhich acts as a ribozyme.
 10. A method according to any one of thepreceding claims, wherein the de novo synthesised nucleic acid issubjected to an amplification step prior to detection.
 11. A methodaccording to claim 10, wherein the amplification step comprises:hybridising the de novo synthesised nucleic acid to a third nucleic acidprobe, which hybridisation forms a second double stranded nucleic acidpromoter either directly, or by 3′ extension of the de novo synthesisednucleic acid using the third probe as template; and adding a polymerasewhich recognises the second promoter so as to cause nucleic acidsynthesis therefrom.
 12. A method according to claim 10, wherein thenucleic acid synthesised from the second promoter is detected.
 13. Amethod according to claim 11, further comprising the steps of:hybridising the nucleic acid synthesised from the second promoter to afourth nucleic acid probe, which hybridisation forms a third doublestranded nucleic acid promoter either directly, or by 3′ extension ofthe nucleic acid synthesised from the second promoter using the fourthprobe as template; and adding a polymerase which recognises the thirdpromoter so as to cause nucleic acid synthesis therefrom.
 14. A methodaccording to claim 13, wherein nucleic acid synthesised from the thirdpromoter is detected.
 15. A method according to claim 13, furthercomprising the step of hybridising nucleic acid synthesised from thethird promoter to the third probe, thereby reforming the second doublestranded promoter, so as to create a cycle of nucleic acid synthesis.16. A method according to claim 10, wherein the amplification stepcomprises: adding third and fourth nucleic acid probes so as to form acomplex comprising the said probes and the de novo synthesised nucleicacid, wherein the third probe comprises the full length sequence of afirst strand of a double stranded promoter, the de novo synthesisednucleic acid comprises an end part of a second strand of the doublestranded promoter which is complementary to a part of the first strand,and the fourth probe comprises the rest of the second strand of thedouble stranded promoter which is complementary to a part of the firststrand, such that a functional promoter is formed when the third probeis hybridised to both the de novo synthesised nucleic acid and to thefourth probe; adding a polymerase which recognises the promoter, so asto cause the synthesis of nucleic acid from the promoter present in thecomplex; and detecting directly or indirectly the synthesised nucleicacid.
 17. A nucleic acid complex comprising three strands of nucleicacid: a promoter strand, a promoter complementary strand, and a targetstrand; wherein the promoter complementary strand comprises the fulllength sequence of a first strand of a double stranded promoter; thetarget strand comprises a part of a second strand of the double strandedpromoter which is complementary to a part of the first strand; and thepromoter strand comprises a part of the second strand of the doublestranded promoter which is complementary to a part of the first strand;wherein neither part of the second strand of the double strandedpromoter present on the target strand or on the promoter strand iscapable of forming a functional promoter when hybridised to the promotercomplementary strand in the absence of the other part, but wherein afunctional promoter is formed when the promoter complementary strand ishybridised to both the target strand and the promoter strand.
 18. Acomplex according to claim 17, wherein the promoter complementary strandand promoter strand are provided by first and second nucleic acid probesrespectively, the complex being formed in performance of a methodaccording to-any one of claims 1-16.
 19. A kit for performing the methodof claim 1, the kit comprising first and second probes for forming,together with the appropriate target sequence, the complex of claim 17,and instructions for performing the method of any one of claims 1-16.20. A kit according to claim 19, further comprising one or more of thefollowing: DNA polymerase; RNA polymerase; ribo- or deoxyribonucleotidetriphosphates; labelling reagents; detection reagents; buffers.
 21. Amethod substantially as hereinbefore described and with reference to theaccompanying drawings.